A trispecific F₂ mapping population was developed by crossing G. arboreum (A₂) cv. Nanking with G. trilobum (D₈), resulting synthetic allotetraploid hybrid 2(A₂D₈). This hybrid was crossed with G. hirsutum (AD₁) cv. T-586 and the resulting hybrid self-pollinated to obtain the segregating F₂ population. The population was used to study inheritance patterns of segregating loci and to establish the linkage groups among the three genome species. A total of 216 markers (194 AFLP, 19 RAPD and 3 morphological markers) were scored, of which 85 markers showed normal Mendelian inheritance. A large number of markers showed distorted segregation perhaps due to a difference of three chromosome arm arrangements between A₂ and AD₁, divergence of the three genome species and areas of low recombination among the genomes. With 2-point linkage analysis, 11 linkage groups were identified that spanning 521.7 cM of the cotton genome with an average distance of 16.8 cM between markers. One morphological marker, pilose, which is located on chromosome 6 was found to be linked with molecular markers in linkage group T1. The addition of more markers will help to identify areas of low recombination among species genome and linkage groups will become more clear and informative.